Angiogenesis, the development and growth of new blood vessels, is important for organ development, wound healing and various pathological conditions such as tumor growth. Angiogenesis involves several processes, such as changes in vascular permeability, as well as endothelial cell adhesion, migration, proliferation and differentiation (Folkman, J. et al., 1996, Cell 87:1153-5). These processes depend both on a number of growth factors as well as on adhesive contacts with the extracellular matrix (ECM) (Breier, G., A. et al., 1997, Thromb. Haemost. 8:678-683, Risau, W., 1997, Nature 386:671-674, Strombald, S. et al., 1996, Chem. Biol. 3:881-885). ECM-associated proteins, such as fibronectin (FN), vitronectin (VN) and fibrinogen (FBG) are deposited into an adhesive fibrillar network and control cellular function including growth, differentiation and migration by transmitting signals to the cells through specific integrins (Giancotti, F. G. et al., 1999, Science 285:1028-32). Observations that mice lacking FN and its receptor α5β1 die early in development and exhibit an improperly formed vasculature indicate that both proteins are crucial participants in physiologic angiogenesis (Fassler, R. et al., 1995, Genes Dev. 9:1896-1908, George, E. L. et al., 1993, Development 119:1079-1091, Yang, J. T. et al., 1993, Development 119:1093-1105). Results of in vitro and in vivo studies provide evidence that FN and αβ1 are critically involved in tumor angiogenesis as well (Hynes, R. O., 2002, Nat. Med. 8:918-921, Kim, S. et al., 2000, Am. J. Pathol. 156:1345-1362).
There is emerging evidence that inflammatory cells, and particularly neutrophils, regulate endothelial cell functions related to angiogenesis. Both pro-angiogenic and anti-angiogenic activities of neutrophils have been described. Neutrophils are a source of growth factors such as the vascular endothelial growth factor (VEGF) and matrix metalloproteinases (Shamamian, P. et al., 2001, J. Cell. Physiol. 189:197-206, Lee, S. et al., 2002, J. Clin. Invest. 110:1105-11; Webb, N. J. et al., 1998, Cytokine. 10:254-7). On the other hand, neutrophil-derived elastase can generate the anti-angiogenic factor angiostatin (Scapini, P. et al., 2002, J. Immunol. 168:5798-804). The net effect of these and potentially other angiogenic products of neutrophils may depend on the biological context, but the manner in which neutrophils regulate angiogenesis in vivo has not been rigorously investigated.
α-defensins, a family of four closely related anti-microbial peptides, are the most abundant proteins found in neutrophil granules, comprising approximately 5% of the total neutrophil protein content (Ganz, T. 2002., J. Clin. Invest. 109:693-697). α-defensins are secreted when neutrophils are activated during phagocytosis of microorganisms or by specific exogenous inflammatory agonists. As small cationic peptides, α-defensins can be incorporated into the cell membrane of prokaryotic organisms during phagocytosis, disrupting ion fluxes and provoking cell lysis (Ganz, T. 2002, J. Clin. Invest. 109:693-697, Kagan, B. L. et al., 1994, Toxicology 87:131-149, Ganz, T. et al., 1985, J. Clin. Invest. 76:1427-35, Harwig, S. S. et al., 1994, Methods Enzymol. 236:160-172). During severe infections, α-defensins are released into the plasma at concentrations approaching 30 μM (whereas such concentrations are normally 15 nM) (Panyutich, A. V. et al., 1993, J. Lab. Clin. Med. 122:202-207). α-defensins accumulate in the vessel wall by binding to ECM-associated FN (Bdeir, K. et al., 1999, Blood 94:2007-2019) and are abundant in human atherosclerotic plaques (Bdeir, K. et al., 1999, Blood 94:2007-2019, Barnathan, E. S. et al., Am. J. Pathol. 150:1009-20); they promote the accumulation of lipoprotein(a) (Higazi, A. A. et al., 1997, Blood 8 9:4290-4298) and inhibit plasminogen activation (Higazi, A. A. et al, 1996, J. Biol. Chem. 271: 17650-17655).
The role of α-defensins in neutrophil biology has heretofore been poorly understood, for example, with respect to neutrophil biology related to tumors. While it is known that tumor vascularization is essential to the growth of tumors in a variety of pathophysiological conditions, it is not known whether α-defensins play any role in neutrophil activity related to tumor biology.
Proliferative retinopathies are major causes of blindness. The prominent feature of these retinopathies is the exuberant neovascularization, which is orchestrated by the hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) that stimulates endothelial cell proliferation, permeability and migration/invasion, as well as by the interaction of extracellular matrix components like fibronectin (FN) with their integrin receptors.
Pathological neovascularization, for example, is a major cause of blindness in infancy and during adulthood, complicating such disorders as the retinopathy of prematurity, sickle cell anemia and diabetes, among others. Neutrophil activation is part of each of these processes, but has been heretofore unknown if α-defensin naturally participates in controlling these processes, or if α-defensin may have any role in the control of these devastating complications.
Because the management and/or inhibition of tumor vascularization is an attractive target for controlling or preventing tumor growth, an understanding of the role of neutrophils, and thereby, α-defensins, in tumor vascularization and related biology is critical to the development of novel anti-angiogenesis therapeutics useful in the treatment of cancer, as well as other diseases, such as retinopathies associated with exuberant and pathological vascular growth. The present invention provides the understanding and goes further to develop therapies and therefore meets these needs.